EDGE (Training network providing cutting-EDGE knowlEDGE on Herpes Virology and Immunology) is a Marie Skłodowska-Curie Innovative Training Network for early stage researchers (ESRs) funded by the European Commission under the H2020 Programme, the EU framework programme for research and innovation. EDGE provides a comprehensive and cross-disciplinary structured curriculum for doctoral students in the fields of virology and immunology. Key research aims of EDGE are to improve the basic understanding of the interactions between herpesviruses and host cells and to uncover implications for the clinical outcome of infections as well as development of vaccines. While each ESR is based in a host institution, there are extensive interactions between laboratories involved, and all positions involve secondments in other laboratories of the network, which also includes a theoretical training program.

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Diseases caused by herpesviruses

The herpesviruses are a family (herpesviridae) of enveloped DNA viruses, of which 9 members are known to infect humans with 5 species being widely spread. Herpes viruses can cause a large spectrum of diseases severity, from mild to potentially lethal. The viruses are highly infective and remain dormant in our bodies after the primary infection. In some cases, reactivation of the virus can occur, causing a secondary infection, which may also be different in nature than the primary disease.

Herpes simplex viruses (HSV-1, HSV-2) are known to infect oral and genital mucosa, respectively. HSV-1 causes gingival, oral and labial herpes (cold sores). The primary disease is represented by vesicular eruption on the lips, oral cavity and gingiva or nose nostrils. The virus is contracted in many cases as early as childhood by human-to-human contact. HSV-2 causes genital herpes – vesicular eruptions on the genitalia (penis, vagina, anus) and is contracted from human to human by vaginal, anal or oral sex. Use of condom during sex can prevent transmission of the virus. Both viruses remain dormant in our nerves and reactivate to form a secondary similar infection. In some cases, primary or secondary infections can occur in the brain and/or its protective layers (meninges), causing severe neurological-infectious diseases – herpes encephalitis and/or herpes meningitis. A special form or herpes meningitis is Mollaret’s meningitis, caused by HSV-2 primarily, leading to a recurrent meningitis in some patients (3-10 episodes throughout life).

Varicella zoster virus (VZV, HHV-3) is the cause of varicella, known as chickenpox, one of the 6 most common rash-causing childhood diseases. The virus causes vesicular eruption all over the body, with distinctive feature of having vesicles in different stages present at the same time. The virus remains dormant in the nerves and its reactivation in older age, then named herpes zoster – painful vesicular eruption in a skin area innervated by the same nerve in which the virus resided (dermatome). VZV can also cause severe encephalitis and meningitis.

Epstein-Bar virus (EBV, HHV-4) is the main cause of the “kissing disease”, infectious mononucleosis. The virus is being transmitted via saliva droplets (coughing, sneezing, sharing cutlery). The virus was recognized as oncogenic (contributing to development of specific cancers).

Human cytomegalovirus (HCMV, HHV-5) is a minor causative agent related to infectious mononucleosis. Moreover, the virus can infect the liver causing transient hepatitis and jaundice (yellowing of the skin and eyes due to accumulation of bilirubin), infection of the lungs or gut, and is a major concern in the very young population and in the immunocompromised patients, such as HIV-infected or those posttransplant.

Human herpesviruses 6 and 7 (HHV-6, HHV-7) can cause mild erythematous skin diseases in young children (erythema infantum, erythema infectiosum), manifested by red skin eruption and high fever. In some cases, the infected child may develop fever seizures that pass after the disease subsided.

Human herpesvirus 8 (HHV-8, KSHV) – was connected to cancerous tumors of the connective tissue (Kaposi’s sarcoma) in immunocompromised patients, mainly in AIDS patients and in some lymphomas.

Transmission of herpesvirus infections

HSV-1 and HSV-2 are transmitted via contact with mucosal and damaged skin surfaces. HSV-1 is mainly tropic for orofacial areas, such as the lips, mouth and nose. In adults, it can be transmitted through saliva during kissing or by sharing food, eating utensils, razors, etc. with an infected person. In some cases, HSV-1 can also cause genital infection through oral-genital contact during sexual intercourse. Although, the risk of transmission increases when there are active sores, virus transmission can occur by healthy skin prior to appearance of ulcers. On the other hand, HSV-2 is mainly tropic for the genital areas. It can be spread through contact with genital surfaces, lesions, sores or fluids of an infected person during sexual intercourse. However, HSV-2 can also cause cold sores in the facial area. In neonates, there is a risk of transmission of HSV if the mother is infected. Prior to, during and shortly after delivery are periods during which the risk of transmission is greatly increased. In addition, mothers with primary HSV infection are more likely to transmit the virus to the neonate.

Transmission of VZV is achieved by direct contact or through the respiratory system via inhalation of virus particles contained in air droplets. The virus mainly infects the respiratory tract (nasopharynx, oropharynx, upper respiratory tract), then leads to viremia, and generalized vesicular lesions.

EBV, the major cause of infectious mononucleosis, is transmitted primarily via direct contact. Exchange of saliva (kissing), sneezing, coughing and sharing food, eating utensils etc. are some practices that facilitate the transmission of the virus from seropositive people. The virus can also be transmitted by blood transfusion.

HCMV is also a cause of mononucleosis, but mainly causes a diverse set of symptoms in immunocompromised patients. The main means of transmission is through saliva, but it can also occur through transfer of infected cells. The most frequent way to transmit HCMV is through sexual intercourse, daily contact between children in school or day-care centres, blood perfusion, organ transplantation, and breast-feeding. Finally, it is important to mention that HCMV is one of the few viruses that can be transmitted from mother to child during pregnancy.

HHV-6, 7 and 8 are mainly transmitted through saliva, where most quantity of these viruses is found, while other modes of transmission are currently under research.

The lytic replication cycle of herpesviruses

The lytic replication of a virus consists in intensive production of viral particles triggering cell lysis and spread of the infection in our organism. The herpesvirus virion is a 150-200 nanometers large particle composed by an external envelope, similar to a cell membrane including viral glycoproteins, a layer of so-called “tegument proteins” and an internal capsid of proteins which directly protects the viral DNA.

The virion attaches to the targeted cell through its surface glycoproteins and enters either by fusion of its envelope with the cell membrane, or by integration of the whole particle into the cytoplasm via an endocytosis mechanism. In the infected cell, the tegument layer is removed and the capsid transported to the nucleus where the viral DNA is released. The herpesvirus genome is transcribed following a sequential and coordinate manner. First, the host replication machinery is hijacked for expression of the viral immediate-early (IE) genes. These IE proteins oppose the immune response and ensure the transcription of the early (E) genes. The E proteins, among which are viral polymerase and helicase, ensure the replication of the viral DNA and help for expression of the late (L) genes. These last encode for the structural proteins of the herpes virion: capsid, tegument and glycoproteins.

Finally, the assembly phase of the new virions starts in the nucleus. Each replicated DNA molecule is encapsidated by one capsid and these so-called “progeny-virions” are translocated in the cytoplasm. There they acquire the tegument layer and the envelope before to reach the cell membrane, be released in the extracellular media and infect the neighbouring cells.

Herpesvirus latency and reactivation

From childhood on, herpesviruses are our life-long companions and while it may all begin with a gentle kiss, herpesviruses can result in painful and often difficult to treat, potentially life-threatening infections. Thus far, 9 herpesviruses have been described to have humans as host, with a prevalence ranging from 50 to more than 90% of the world population. After a primary infection, viral replication is usually controlled by the immune system although the viruses are not cleared from the human host. Instead they establish latent infections in specific cell types, and remain hidden from immune surveillance, as they silence almost completely any expression of their genes. During latency, the immune system remains on alert, and contributes to the viruses’ mechanisms to maintain their genomes in post-mitotic cells, or to replicate the viral genomes prior to cell division. For this purpose, different herpesviruses have evolved sophisticated latency programs that are controlled by viral proteins and viral miRNAs.

Unfortunately, some conditions can weaken the immune surveillance in favour of the virus’ reactivation and replication, leading to a spread of the viral infection within the host. This is particularly the case in children, elderly people, HIV infected patients and transplant recipients. Reactivation of viral replication can result in brain damage, hearing loss, vision impairment, graft rejection and death, if left untreated. Thus far, there is no treatment to eliminate of the latent viral genomes from the body, and with the exception of varicella zoster virus, there are also no prophylactic vaccines. Present treatment regimens can only control virus replication and dissemination, therefore the risk of reactivation and emergence of resistant strains remain.

Treatment of herpesvirus infections

In general, herpesviral infections are treated with different antiviral drugs to reduce viral shedding but due to the latency phase of herpesviruses, patients can experience reactivation of infection causing recurrent disease. The treatment and drug of choice depends on the type of herpesvirus and the individual patient. For the treatment, acute infections and recurrence of latent viruses are differentiated as well as the immune status of the patient. Immunosuppressed patients are more prone to severe diseases associated with herpesvirus infections. Systemic drugs including acyclovir, famciclovir and valacyclovir showed clinical benefit for Herpes simplex virus type 1 (HSV-1) and HSV-2, and to a lesser degree against Varicella-zoster virus (VZV) - infected patients. Other systemic antiviral drugs including ganciclovir and valganciclovir are also used for HSV-1 and HSV-2 as well as for Cytomegalovirus (CMV). The drug foscarnet proved beneficial against acyclovir and ganciclovir-resistant herpesvirus infections. However, these treatments do not affect latent virus or viral recurrences. Chicken pox caused by VZV can be prevented with the chickenpox vaccine that proved very safe and effective in clinical studies. Moreover, a vaccine against zoster (shingles) has been developed for the elderly age-group. These are the only examples for a herpesvirus with an existing vaccine available. Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) are the only examples for cancer-associated human herpesviruses. No specific antiviral drugs exist and the treatment focuses on the symptoms and the cancer treatment including surgery, chemotherapies and local irradiations.

A lot of effort to design specific treatments is invested and several clinical studies are currently testing new antiviral drugs against herpesviruses.

Want to learn more about the immune system?

Introduction

The immune system is our fundamental defense mechanism. It has aided the evolution of our species, enabling us protection from pathogens such as bacteria, viruses and fungi. Without this complex system of immune cells and defense proteins, we would quickly succumb to lethal infections.

The immune system has an incredibly intricate job of identifying invading microbes and to distinguish self from non-self. This is a fine balance between the recognition of dangerous invaders such as E.coli bacteria or Influenza virus, and the identification of our own cells and proteins. For most humans, our immune system functions in a way that offers us protection from pathogens that might want to do us harm. However, the immune system can also be the very thing that causes harm if it malfunctions, resulting in autoimmune diseases like sclerosis and arthritis.

Even a fully functional immune system is also not always able to eliminate infections, as seen for instance for herpesvirus infections. This is because pathogens have evolved counter measures which enable them to subvert the immune system. This prevents our defense mechanism from identifying and attacking potentially disease-causing microorganisms. Given the enormous impact of the immune system on human health, there is a need to further understand this complex system. Research in immunology may lead to new vaccines and treatments against infections, inflammatory diseases, and cancer.

The innate immune system and herpesviruses

From an evolutionary perspective, the innate immune system is older than the adaptive immune system. Parts of the innate immune system can be found in species that arose long before humans populated the earth. At the same time, herpesviruses have existed for around 200 million years while mammals only started to arise around 80 million years ago. Consequently, for the longest time have the ancestors of the modern human co-existed and co-evolved with herpesviruses. This includes the evolution of innate defence mechanisms against herpesviral infections in humans, as well as the development of innate immune evasion mechanisms by herpesviruses.

The significance of the innate immune system, in the context of herpesvirus infections, is most drastically illustrated by individuals that suffer from relatively uncommon, life-threatening complications of an infection. At least five out of nine human herpesviruses infect major parts of the population without causing acute and severe complications upon infection. Individuals that suffer from complications, like severe herpesviral encephalitis, frequently have genetic mutations that render integral parts of the cell-intrinsic, innate immune response non-functional. This includes, for example, genetic defects in so-called “pattern-recognition receptors”, molecules that normally detect virus presence and initiate cellular defence responses. Furthermore, certain primary immunodeficiencies predispose affected individuals selectively to acute herpesviral infections. This demonstrates the non-redundant function of the innate immune system, in particular the cell-intrinsic part of it, in the defence against herpesviruses.

The adaptive immune system and herpesviral infections

Most infections are controlled by the innate immune system. However, if pathogens overcome this first line of defense, the adaptive immune system will be mobilized and both systems synergize to eliminate pathogens. In contrast to the immediate activation of the innate immune system, the response time of the adaptive immune system can take several days. The adaptive immune system is comprised by B- and T-lymphocytes. B-cells produce antibodies that specifically bind to foreign antigens and mark invaders for destruction. T-cells either differentiate into cells that participate in lymphocyte maturation and regulation or kill virus-infected cells.

One key feature of the adaptive immune system is its memory function, carried out by memory cells that can last for years in the human body. Thus, recurrent infections by the same pathogen can be easily recognized and trigger highly specific responses that eliminate the pathogen in most cases.

With this knowledge, one would assume that pathogens, including herpesviruses, could be easily fended off. However, through a long-period of co-evolution with their host, herpesviruses have acquired dedicated immune evasion proteins. These immune evasion proteins specifically interfere with critical steps during the adaptive immune response, thus allowing the virus to maintain/establish a lifelong infection.

Evasion of the immune system by herpesviruses

Upon infection with herpesviruses, a strong immune response is induced. Therefore, through millions of years of coevolution with the host, herpesviruses had to evolve several effective strategies to counteract different parts of the host immune response. This was a prerequisite for their ability to establish lifelong persistence in their hosts.

Herpesviruses need to defeat different lines of defence in order to invade and replicate in their host. In this battle, they encounter upon entry a first row of “soldiers”, resembling the innate immune system. This host defence acts immediately to prevent virus replication and, at the same time, warns the second row of “soldiers”, the adaptive immune system. This second line of defence then develops a specific attack tailored to the pathogen that is trying to intrude.

Herpesviruses have evolved a variety of sophisticated mechanisms to deal with this potent immune response: They are able to hide while they enter, and are therefore invisible for the “soldiers”. Another strategy is to switch off certain control points, or they even use the host response for their own benefit. Specific examples are that they can inhibit the innate immune system by preventing the destruction of infected cells by so-called natural killer cells, or the antiviral type I interferon response, which usually is key to develop an antiviral state in the infected cell. They are also able to inhibit the adaptive immune response mediated by MHC class I antigen processing and presentation, which leads to a reduction in T cell recognition and T cell-mediated killing of infected cells.

By subverting multiple facets of the host immune response, these viruses are thus not eliminated and can secure lifelong infection of their host.

Herpesvirus infections in immunodeficient individuals

Immunodeficient individuals are defined as having a missing or defective part of their immune system. As such, they are unable to mount an effective immune response to a foreign invader, making them more vulnerable to severe infections even by microorganisms that are not known to cause severe diseases in a healthy, immunocompetent individual. This can either be a primary immunodeficiency, that is inherited, or secondary, which is acquired throughout a person’s life. In recent years, increasing evidence describing inherited predispositions to a specific infection have emerged, including in the field of herpesviruses. Herpes simplex encephalitis (HSE) has been linked to mutations in the Toll-like receptor 3, which is key in our cells ability to recognise viral infection. Furthermore, primary immunodeficiencies have also been described in children with central nervous system (CNS) complications following varicella zoster virus (VZV) infection, due a defect in the viral DNA sensor RNA Polymerase III (Pol III).Also key to an individual’s ability to fight an infection are immune cells known as natural killer (NK) cells. Patients with a NK cell deficiency have an increased susceptibility to herpesviruses, and Epstein-Barr virus (EBV) infection is potentially fatal in patients with the inherited X-linked NK cell immunodeficiency called Duncan syndrome. Furthermore, immunodeficiencies can be acquired, rather than inherited. Most well described is the immunocompromised status of patients following infection with the human immunodeficiency virus (HIV). Herpesviruses can take advantage of this, and re-activate to causes a severe disease. Furthermore, patients under immunosuppressive therapy for autoimmune diseases or after organ transplantation can all render an individual immunodeficient, transiently. In such patients, opportunistic infection with human cytomegalovirus (CMV) can cause severe disease such as pneumonitis. Moreover, human herpesvirus-8 or Kaposi’s sarcoma-associated virus can cause tumour formation in the skin and mouth of individuals with a weakened immune system. Treatment of secondary immunodeficiencies often includes anti-viral drugs to treat opportunistic infection such as those caused by herpesviruses, antibiotics to prevent and treat bacterial infections and immunoglobulin treatment, but also involves controlling or treating the underlying cause, such as HIV. In the case of inherited primary immunodeficiencies, treatment may include long-term prophylactic anti-viral or immune-boosting therapies.

Potential roles of herpesviruses in autoinflammatory diseases

An autoinflammatory disease is characterized by a state of chronic inflammation due to an abnormal over activity of the immune system against the body’s own tissues. Genetic predisposition and environmental factors, such as an exposure to particular chemicals or infections, play a major role in the disease development.

Viral infections boost a strong immune response that is necessary for the viral clearance; in some circumstances the immune system goes out of control and overreact attacking not only the viral particles but also the host itself. Several members of the Herpesviridae family have been associated with autoinflammatory diseases, for example infection with Epstein-Barr virus might increase the risk to develop multiple sclerosis, characterized by the formation of lesions and inflammation in the central nervous system. Herpes simplex virus (type I and/or II) causes herpetic stromal keratitis which leads to stromal scarring and vision loss. Out of all herpesviruses, human cytomegalovirus has been linked to multiple chronic conditions such as diabetes, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, psoriasis and Sjögren’s syndrome.

It is not known why almost every member of this family has been related to one or more AIDs, but the one could speculate that the ability of these viruses to establish a lifelong infection and to manipulate the immune system might be relevant in this contest.

Challenges and opportunities

Despite available drugs against some herpesviruses there are still insufficient options for treatment and prevention of infections with these viruses. With the recent advance in the understanding of immune response to infections, there are new opportunities to use this knowledge.

Development of vaccines against herpesviruses

There are a few effective antiviral agents available for the treatment of herpes simplex (HSV-1) and genital herpes (HSV-2), but there are no vaccines against herpesviruses currently on the market.

Both prophylactic and therapeutic HSV-vaccine platforms are being investigated, such as inactivated HSV-2 viruses and protein subunit vaccines with focus on one of the major surface antigen of the virus, Glycoprotein D (gD2). Furthermore, several live-attenuated or replication-defective virus vaccine candidates are also being explored. Yet, these vaccines tend to exhibit more safety concerns.

The development of a vaccine in the preclinical phase is performed in animal models. Mice are commonly used (Dagsputa et al., 2011) but guinea pigs are for example preferred as HSV-2 infection model as they mimic human HSV-2 infection better and show spontaneous genital reactivation (Stanberry et al., 1982). Lately, rhesus macaques have also been exploited (Awasthi et al., 2017).Both humoral and cellular immune responses have been shown to be essential for protection in animal models, but lately considerable attention has been shifted to mucosal immune responses based both on antibodies and tissue resident T-cells.

Research continues as the gD2 subunit vaccine Herpevac failed to protect against HSV-2 in clinical trials, despite showing efficacy in pre-clinical models. Post hoc analysis revealed that gD neutralizing antibody titers did not correlate with HSV-2 protection, emphasizing the importance of thoroughly investigating potential correlates of protection during the development of vaccines.

Development of new antiviral drugs

Herpesvirus infections are life long, and so far cannot be clinically cured, in the sense that the latent viral genomes cannot be eliminated from an infected host. Clinical symptoms are managed by administration of drugs that target the replication cycle of the virus – antiviral compounds. Treatment with antivirals can be used upon virus manifestation or in a preventive manner in case of severe immunedeficiencies.

Acyclovir and its derivatives are the most active antiviral compounds against herpes simplex type 1 and type 2, but are less effective against other herpesviruses, e.g. Varicella zoster virus or Epstein Barr virus. Non-infected cells do not incorporate Acyclovir into their replicated genomes, while viral enzymes activate the compounds, and cease viral DNA replication. However, similarly to antibiotics and resistant bacteria, herpesviruses can also acquire resistance to antiviral medication upon mutation. There is an urgent need to develop additional drugs to allow combinatorial antiviral therapy against diseases caused by the various herpesviruses.

Therefore, many labs worldwide, including those of EDGE, have development medium and high-throughput cellular infection assays to screen various libraries of small synthesized chemical compounds and natural microbial products with the aim to identify potential antiviral components to be developed into novel antiviral therapy. Furthermore, small peptides are also tested for their antiviral effects. Upon testing these compounds in small animal infection models, new solutions may arise to relieve the social-economic burden caused by herpesvirus diseases.

Elimination of latent herpesvirus infections – is it possible?

Herpesviruses infect the majority of the human population and can cause significant morbidity and mortality. Herpesviruses are characterized by their ability to undergo a productive infection upon infection of the host and then spread to establish a latent infection allowing them to persist in the host for life.

During the latent stage of infection, the virus is present in cells but limits its gene expression and DNA replication to a minimum. As such, the virus can efficiently evade the host immune response. Specific stimuli can lead to a reactivation of the virus leading to virus replication and release of progeny virus that can infect other cells and individuals.

Current treatment options to restrict the clinical manifestations of herpesvirus infections are limited to agents that target the productive, but not the latent, phase of infection. These drugs can block virus replication, but do not eliminate the virus from the host. As such, there is currently no cure from a herpesvirus infection.

The recent development of new molecular tools, e.g. using CRISPR/Cas9 genome engineering techniques, enables genetic targeting of viral DNA. One research group in the ITN EDGE consortium showed the utilization of this method to impair viral replication and clear latent virus infections in herpesvirus infected cells. These findings offer new ways in targeting latency of herpesviruses and open up new perspectives for the development of future treatment options.

Prevention of herpesvirus diseases in the elderly

Over the last half century, the increasing life expectancy worldwide has resulted in the growth of the population over 50 years old. Although this population will continue to increase in size, it may not age healthily. As a result of immunosenescence (the gradual decline in the ability of the immune system to fight infections), co-morbidity and general frailty, the elderly are more susceptible to infectious diseases. Moreover, these individuals are more prone to infections not only with emergent pathogens, but also with infections they have encountered previously.

Herpesviruses are prevalent among elderly populations. The five causing the most health concerns are: herpes simplex virus (HSV) type 1 and 2, Epstein–Barr virus (EBV), cytomegalovirus (CMV), and varicella zoster virus (VZV). These well adapted pathogens establish a latent lifelong infection, manipulate the host immune system in a variety of ways, and may reemerge after many decades at unpredictable times to cause severe complications. For example, herpes zoster (HZ or shingles) strikes millions of older adults annually worldwide and disables a substantial number of them via postherpetic neuralgia (PHN).

Vaccinating people aged 50 and older against herpesviruses may be one strategy to promote healthy aging. At present, only one vaccine is licensed for the prevention of herpesvirus infection, a live attenuated vaccine against VZV, which is approved by the FDA. The administration of passive immunization may also prevent, or at least attenuate, shingles in high-risk individuals. Currently, experimental vaccines for Epstein-Barr, HSV-1 and 2, CMV are in various stages of clinical trials.